Timing Is Everything in Studying Disorderly Proteins

Scientists are developing methods to catch these molecules in the act of folding and transitioning.

Treating human diseases caused by misfolded proteins requires scientists to identify the potentially pathogenic folded protein. Most methods for studying proteins have been designed to detect conformationally stable proteins, however. Developing analytical methods to predict how a protein will fold under a given set of conditions or catch them in transient states has proven challenging.

To date predicting how chains of amino acids fold has required either powerful supercomputers or cloud sourcing that uses the pattern recognition power of thousands of people by means of games such as Folding@home. The program, funded by the NIH and NSF and based at Stanford University and Stanford Medical School, studies protein folding, misfolding, aggregation, and related diseases.

The project reportedly has been able to simulate time scales thousands to millions of times longer than was previously possible. It has allowed the simulation of folding for the first time and the examination of protein folding-related disease.

Computer model-based prediction, however, can be time consuming and often inaccurate. Some scientists say that the protein-structure bottleneck has slowed the utilization of DNA sequence data for medical and biotechnology applications. Researchers are working on developing multimodal methods that can catch individual proteins in the act of folding and in physiological context to help characterize the consequences of misfolding.

Coupling Single-Molecule FRET and Microfluidic Mixing

Scientists at The Scripps Research Institute and the University of California, San Diego (UCSD) recently reported on a new technique that enables detection in less than 0.001 seconds of transiently folded single-molecule structures from a class of rapidly shape-shifting molecules known as intrinsically disordered proteins (IDPs). The method, described in Nature Methods, also permits new types of observations of short-lived protein complexes and allows analysis of IDPs associated with human neurodegenerative disorders, including the α-synuclein protein associated with Alziehmer and Parkinson diseases.

Unlike proteins with a well-defined 3-D structure that determines their cellular functions, IDPs have long stretches of sequence that are relatively devoid of structure. These protein systems are very prevalent in genomes, especially in more evolved organisms. The physics of these systems may play a key role in many cellular functions including signaling, scaffolding, gating, and timing, according to Ashok A. Deniz, Ph.D., associate professor, department of molecular biology, California campus of Scripps.

To address analyses of these proteins, Dr. Deniz and co-researchers Alex Groisma, Ph.D., associate professor of physics at UCSD, and Yann Gambin, Ph.D., of Scripps, combined and improved upon two established experimental methods: single-molecule Förster Resonance energy transfer (smFRET) and microfluidic mixing.

“Fast laminar microfluidic flows have been used for fast mixing, but under these conditions, the molecules go by the detectors too fast to collect enough photons for good signal-to-noise for smFRET measurements,” Dr. Deniz told GEN.

“Hence, previous smFRET measurements used slower flows resulting in slower mixing. In our device, by combining fast mixing in fast flow with rapid deceleration just prior to detection, we could then collect enough photons for smFRET analysis very soon after a change in conditions.”

Dr. Deniz told GEN that understanding the structural properties and complicated dynamics of intrinsically disordered proteins is difficult with many conventional ensemble experiments that average properties of millions of molecules. Commenting on the microfluidics used in his approach, he said, “We reported a method in which you can look at changes in structural distributions following a rapid change in conditions—in the case of α-synuclein, the presence of a binding partner—to see what happens to the distribution as a function of time.”

“We have pushed the time resolution to below a millisecond for microfluidic single-molecule FRET under nonequilibrium conditions. And not only can we change the conditions surrounding the protein, but we can also dilute the protein solution rapidly, which is very useful for looking at relatively weak complexes that would normally dissociate under single protein conditions. Now we can watch them for a while before they dissociate.”

Leveraging a similar technique to Dr. Deniz’, physicists working at UC Santa Barbara (UCSB) in collaboration with colleagues at the University of Zurich made the first subsecond, single-molecule measurements of a chaperonin protein. Their study, published in the June 2010 issue of the Proceedings of the National Academy of Sciences, used a microfabricated fluid mixing device built at UCSB’s nanofabrication facility.

Chaperonins comprise a group of proteins that help other proteins fold correctly and prevent protein aggregation. GroEL, one member of this protein family, works with GroES to encapsulate a substrate protein, isolating it as it folds in the cell. Inside the fluid mixing device the scientists followed the folding of the protein rhodanase inside the GroEL/GroES chaperonin molecular cavity over a time range from milliseconds to hours.

Fast Relaxation Imaging

No matter what method is used, researchers studying disorderly proteins agree that time is of the essence for catching proteins in action. To study the biomolecular dynamics inside of a single living cell, Martin Gruebele, Ph.D., at the University of Illinois’ department of chemistry, and his team developed a method they call fast relaxation imaging, or FreI. Details of the technique were published in Nature Methods during February 2010.

The scientists used FreI, which combines fluorescence microscopy and temperature jumps, to probe biomolecular dynamics and stability inside single living cells with high spatiotemporal resolution. By measuring the reversible fast folding kinetics as well as folding thermodynamics of a FRET probe-labeled phosphoglycerate kinase construct in two human cell lines, the scientists showed that the cell environment influences protein stability and folding rate.

“It’s a tool that combines two worlds: chemical dynamics and the ability to study reactions as they occur with biological environments, where cell biologists observe how reactions occur in cells,” Dr. Gruebele explained.

To achieve both a fast upward and downward temperature spike in the cells, the investigators used programmed laser pulses to preheat, spike, plateau, and finally stabilize the temperature in the cell and its aqueous medium. An inverted fluorescence microscope was used to observe and record the happenings inside the cell, all of which occur over a few milliseconds. “This is the first experiment that allows us to observe the dynamics of a protein folding in a live cell,” Dr. Gruebele said.

Furthermore, the technique provides a means of looking at how fast biological processes occur as a function of time, Dr. Gruebele added. For example, he explained, “we can take these proteins that cause these diseases, actually put them into the kind of cells where they cause these diseases, give them a heat shock, and actually see if they bind differently to the membranes, if they cause the membrane to puncture. We’ll be able to follow these events in real time and give researchers an idea of if this is a possible pathway through which disease could occur.”

Several control mechanisms regulate the various stages of protein synthesis and trafficking in the cell. Both factors help maintain the conditions for proper folding. These processes also ensure that improperly folded or damaged proteins are degraded before they can accumulate to form toxic complexes.

The techniques discussed in this article and others in development open a new window to mechanistic understanding of how misfolded proteins may contribute to human disease. Novel and enhanced multimodal methods to watch proteins change under conditions that can be manipulated will improve the odds of new drug target identification. These targets will likely include misfolded proteins themselves as well as other molecules that regulate their movement through the cell and will be discussed in part two of this article.

Jobs

GEN Jobs powered by HireLifeScience.com connects you directly to employers in pharma, biotech, and the life sciences. View 40 to 50 fresh job postings daily or search for employment opportunities including those in R&D, clinical research, QA/QC, biomanufacturing, and regulatory affairs.

Be sure to take the GEN Poll

Climate Change

How would you describe the researchers response to the 2°C global temperature target?

They are right on the mark. A 2°C global temperature rise will push us further over the edge. We have to set a lower target.

A 2°C global temperature rise is a reasonable target on which to focus in order to diminish the impact of climate change.

We have already gone beyond the threshold for getting a handle on climate change. So now we just need to learn how to adapt to the inexorable climatic changes with which we will have to deal going forward.

They are right on the mark. A 2°C global temperature rise will push us further over the edge. We have to set a lower target.

45.5%

A 2°C global temperature rise is a reasonable target on which to focus in order to diminish the impact of climate change.

9.1%

We have already gone beyond the threshold for getting a handle on climate change. So now we just need to learn how to adapt to the inexorable climatic changes with which we will have to deal going forward.

If you have any questions about your subscription, click
hereto email us or call at (914) 740-2189.

You may also be interested in subscribing to the GEN magazine, an indispensable
resource for everyone involved in the business of translating discoveries at the
bench into solutions that fight disease and improve health, agriculture, and the
environment. Subscribe
today to see why over 60,000 biotech professionals read GEN to
keep current in the areas of genomics, proteomics, drug discovery, biomarker discovery,
bioprocessing, molecular diagnostics, collaborations, biotech business trends, and
more.